xe2x80x9cMicroarraysxe2x80x9d are devices used in biotechnology and other science research, and can be made by putting a large number of tiny droplets of DNA (or other target such as cDNA or proteins) on a glass slide. Short pieces of DNA, called probes, are then applied to the DNAs on the slide. Typically, the probes are fluorescent, so they light up when short wavelength light is shone on them (the probes can also be labeled with other substances to reflect or otherwise emanate light when they are scanned). Microarrays can be used, for example, to study how large numbers of genes interact with each other (genes are made of DNA), or how a cell is able to simultaneously control vast numbers of genes.
The probes stick to the microarray wherever the probes find target stretches of DNA called complementary DNA strands. The microarrays are then put into a scanning microarray reader that measures the brightness of each fluorescent dot: the brighter the dot, the more probe (and thus the more target DNA or other biological material) is present. This can indicate, for example, how active the target is, or where it is on the slide.
Microarrays can be used, for example, to study genomic content, how large numbers of genes interact with each other (genes are made of DNA), or how a cell is able to simultaneously control vast numbers of genes (expression patterns). Different types of microarrays include, but are not limited to, cDNA arrays, oligonucleotide arrays and protein arrays.
Some general concepts about microarrays, and the microarray readers that measure the dots, are discussed in more scientific terms in the following paragraphs.
Fluorescence based microarray readers suffer from limited dynamic range with respect to the possible intensities of target spots in, for example, cDNA expression microarrays. Systems which use scanning spots and photomultiplier tubes (PMTs) for detection are reported to possibly have a xcx9c106 dynamic range but may have a 104 to 105 dynamic range in practice. Most charge couple device (CCD) imaging microarray readers have about a xcx9c103 dynamic range (12 bit-digitization, 4096 levels, 40,000-1,000,000 electron well depths, 200:1 up to 1000:1 possible signal-to-noise ratio).
The intensity of a fluorescent target spot on a microarray is a function of factors such as how long the target spot is sampled (data sampling time, which can be dwell time for scanning spot systems and integration time for CCD imaging systems), the intensity of the illumination (illumination intensity), the sensitivity of the detector (quantum efficiency, signal transducer, or measurement sensitivity), and the accuracy of digitization (pulse counting or voltage digitization). Coordination and control of these factors is difficult, so measuring spot intensity over a wide range is difficult.
Accordingly, there has gone unmet a need for improved methods of precisely measuring the brightness of the target spots on a microarray over a wide range of target spot intensity. The present invention provides this and other advantages.
Target spots on a microarray that are too dim or too bright for the microarray reader to accurately measure are a problem, for example because they fall outside of certain threshold levels so the microarray reader cannot accurately measure them, or because target spots that are too bright can also hinder the measurement of neighboring target spots due to glare or other interference. Typically, the intensity of light emanating from target spots is proportional to the amount of light shown or incident on the target spots; the more light that is incident on the target spot (excitation or illumination light), the brighter the light coming from the target spot. The present invention takes advantage of this and adjusts the amount of light directed at specific non-acceptable target spots (for example, those spots which fall outside the dynamic range of the system in use), such that dim spots receive more excitation light and overly bright spots receive less.
Similarly, in conjunction with or instead of such actions, the present invention adjusts the amount of light received from specific non-acceptable target spots, such that the detector receives more light from dim spots and less light from overly bright spots. This increases or decreases, respectively, their measured brightness, which in turn effectively increases the range over which a microarray reader can accurately measure the spots, and can also improve the signal-to-noise ratio and other aspects of the measurements. In some embodiments, the present invention can increase the range of the microarray reader by up to about 1000 times or more, and improve the signal-to-noise ratio for target spots up to about 16 times or more.
In one aspect, the present invention provides automated methods of reading a microarray comprising, a) providing an initial representation of a microarray comprising a plurality of target spots illuminated by illumination light having a designated intensity; b) determining from the initial representation whether at least one of the target spots has an emanating light intensity that can be not between selected upper and lower threshold values, and designating such target spot a non-acceptable target spot; and, c) modulating the designated intensity of the illumination light via an automated upstream selective light modulator located in an illumination light path substantially at a conjugate image plane of the sample to provide a modulated illumination light and an adjusted target spot that emanates an adjusted light intensity between the selected upper and lower threshold values.
In some embodiments, the methods further comprise measuring the amount of modulation of the designated intensity of the illumination light and measuring the adjusted light intensity, then correlating the amount of modulation with the adjusted light intensity to provide a measure of the actual signal strength of the target spot. In this and other embodiments of the invention (unless expressly stated otherwise or clear from the context), all embodiments, aspects, features, etc., of the present invention can be mixed and matched, combined and permuted in any desired manner. The methods can further comprise determining an amount of a probe located at the adjusted target spot from the measure of the actual signal strength of the target spot. The methods are suitable for detecting any light emanating spot, such as reflective, fluorescent or other light.
The methods can be implemented according to various formulae. Such formulae include:
SS(x,y)=K*CCDS(x,y)/II(x,y) xe2x80x83xe2x80x83(1) 
where,
SS(x,y) can be the actual signal strength of the target spot,
K can be a constant for the system,
(CCDS(x,y)) can be the adjusted light intensity, and
(II(x,y)) can be the modulated illumination light.
SS(x,y)=K*PB(II(x,y),fluoro)*CCDS(x,y)/II(x,y) xe2x80x83xe2x80x83(2) 
where,
SS(x,y) can be the actual signal strength of the target spot,
K can be a constant for the system,
PB(II(x,y),fluoro) can be a photobleaching function based on illumination energy/intensity and a fluorophore being excited,
(CCDS(x,y)) can be the adjusted light intensity, and
(II(x,y)) can be the modulated illumination light.
SS(x,y)=K*PB(II(x,y),fluoro,x,y)*CCDS(x,y)/II(x,y) xe2x80x83xe2x80x83(3) 
where,
SS(x,y) can be the actual signal strength of the target spot,
K can be a constant for the system,
PB(II(x,y),fluoro,x,y) can be a photobleaching function based on illumination energy/intensity, a fluorophore being excited, and a spatial variation term,
(CCDS(x,y)) can be the adjusted light intensity, and
(II(x,y)) can be the modulated illumination light.
The modulated illumination light can be modulated by changing its illumination intensity, by changing its duration of illuminating the target spot, or otherwise as desired. The initial representation can comprise a precompiled map of expected data for the target spots of the microarray, or an initial image of the plurality of target spots illuminated by the illumination light having the designated intensity, typically taken by a same microarray reader that implements other elements of the methods. The initial image can be taken substantially immediately before the determining, modulating, measuring and correlating are implemented.
The methods can further comprise repeating any desired element, such as the determining, modulating, measuring and correlating in an iterative fashion, for example using the measure of the actual signal strength as the initial representation. The probe can be selected such that the modulation is linearly related to the adjusted light intensity.
In certain embodiments, the methods can be implemented using a microarray reader comprising the upstream selective light modulator, and a light detector disposed downstream from the microarray in a detection light path substantially at a conjugate image plane of the sample, wherein the selective light modulator and the light detector can be operably connected to at least one controller containing computer-implemented programming that controls transmissive characteristics of the upstream selective light modulator and that compiles the modulated illumination light and the adjusted light intensity, and wherein the controller spatially varies the transmissive characteristics of the selective light modulator to vary the modulated illumination light impinging on the non-acceptable target spots of the microarray such that light emanating from the non-acceptable target spots can be between the threshold levels. The upstream selective light modulator can comprise a digital micromirror device and the detector can comprise a charge coupled device.
In other aspects, the present invention provides automated methods of reading a microarray comprising, a) providing an initial representation of a microarray comprising a plurality of target spots illuminated by illumination light having a designated intensity; b) determining from the initial representation whether at least one of the target spots has an emanating light intensity that can be not between selected upper and lower threshold values, and designating at least one of such target spots a non-acceptable target spot; and, c) modulating the emanating light intensity via an automated downstream selective light modulator located in a detection light path substantially at a conjugate image plane of the sample to provide a modulated detection light comprising an adjusted emanating light intensity that can be between the selected upper and lower threshold values.
The methods can further comprise measuring the amount of modulation of the detection light and measuring the modulated detection light, then correlating the amount of modulation with the modulated detection light to provide a measure of the actual signal strength of the target spot. The methods can also further comprise determining an amount of a probe located at the non-acceptable target spot from the measure of the actual signal strength of the target spot.
Such methods can be implemented using a microarray reader comprising the selective light modulator, and a light detector disposed in a detection light path substantially at a conjugate image plane of the sample and downstream from the microarray and the downstream selective light modulator, wherein the selective light modulator and the light detector can be operably connected to at least one controller containing computer-implemented programming that controls transmissive characteristics of the downstream selective light modulator and that compiles the modulated detection light and the adjusted light intensity, and wherein the controller selectively varies the transmissive characteristics of the selective light modulator to vary the modulated detection light impinging on the non-acceptable target spots of the microarray such that light received at the detector can be between the threshold levels.
In a further aspect, the present invention provides microarray reader comprising an automated upstream selective light modulator located upstream of a microarray in an illumination light path substantially at a conjugate image plane of the sample, and a light detector disposed downstream from the microarray in a detection light path substantially at a conjugate image plane of the sample, wherein the selective light modulator and the light detector can be operably connected to at least one controller containing computer-implemented programming that controls transmissive characteristics of the upstream selective light modulator and that compiles an amount of modulated illumination light when the upstream selective light modulator can be modulated and an adjusted light intensity emanating from a target spot on a microarray receiving the modulated illumination light, and wherein the controller selectively varies the transmissive characteristics of the selective light modulator to vary the modulated illumination light impinging on at least one non-acceptable target spot of the microarray such that light emanating from the at least one non-acceptable target spot can be between selected threshold levels.
The controller can further comprise computer-implemented programming that implements other aspects of the methods discussed herein. For example, the programming can control measuring the amount of modulation of the illumination light and control measuring the adjusted light intensity, then correlate the amount of modulation with the adjusted light intensity to provide a measure of the actual signal strength of the target spot. The programming can determine an amount of a probe located at the at least one non-acceptable target spot from the measure of the actual signal strength of the target spot.
In another aspect, the present invention provides microarray reader comprising an automated downstream selective light modulator located downstream of a microarray in a detection light path substantially at a conjugate image plane of the sample, and a light detector disposed in the detection light path substantially at a conjugate image plane of the sample and downstream from the downstream selective light modulator and the microarray, wherein the downstream selective light modulator and the light detector can be operably connected to at least one controller containing computer-implemented programming that controls transmissive characteristics of the downstream selective light modulator and that compiles an amount of modulated detection light when the downstream selective light modulator can be modulated and an adjusted light intensity received by the detector, and wherein the controller selectively varies the transmissive characteristics of the downstream selective light modulator to vary the modulated detection light emanating from at least one non-acceptable target spot of the microarray such that light received at the detector from the at least one non-acceptable target spot can be between selected threshold levels.
As above, the controller can further comprise programming that implements other features of the methods. The controller can control measuring the amount of modulation of the detection light and control measuring the adjusted light intensity, then correlate the amount of modulation with the adjusted light intensity to provide a measure of the actual signal strength of the target spot. The controller can also determine an amount of a probe located at the at least one non-acceptable target spot from the measure of the actual signal strength of the target spot.
In still other aspects, the present invention includes automated methods of reading a microarray comprising, a) providing an initial representation of a microarray comprising a plurality of target spots illuminated by illumination light having a designated intensity; b) determining from the initial representation whether at least one of the target spots has an emanating light intensity that can be not between selected upper and lower threshold values, and designating at least one of such target spots as a non-acceptable target spot; c) selectively illuminating the non-acceptable target spot via selectively transmitting light to the microarray using a first automated upstream selective light modulator located in an illumination light path substantially at a conjugate image plane of the sample; and, d) modulating the designated intensity of the illumination light via a second automated upstream selective light modulator located in the illumination light path substantially at a conjugate image plane of an aperture diaphragm of the objective lens, to provide a modulated illumination light and an adjusted target spot that emanates an adjusted light intensity between the selected upper and lower threshold values.
The methods can also modulate the designated intensity of the illumination light via the first automated upstream selective light modulator located in the illumination light path substantially at the conjugate image plane of the sample, and can determine an amount of a probe located at the adjusted target spot.
In yet further aspects, the present invention comprises automated methods of reading a microarray comprising, a) providing an initial representation of a microarray comprising a plurality of target spots illuminated by illumination light having a designated intensity; b) determining from the initial representation whether at least one of the target spots has an emanating light intensity that can be not between selected upper and lower threshold values, and designating at least one of such target spots as a non-acceptable target spot; c) selectively detecting light from the non-acceptable target spot via selectively transmitting light from the microarray using a first automated downstream selective light modulator located in a detection light path substantially at a conjugate image plane of the sample; and, d) modulating the emanating light intensity via a second automated downstream selective light modulator located in a detection light path substantially at a conjugate image plane of an aperture diaphragm of the objective lens, to provide a modulated detection light comprising an adjusted emanating light intensity between the selected upper and lower threshold values.
The methods can further comprise also modulating the emanating light intensity of the detection light via the first automated downstream selective light modulator located in the detection light path substantially at the conjugate image plane of the sample.
In additional aspects, the present invention includes microarray readers configured to implement such methods. For example, the readers can comprise a first automated upstream selective light modulator located upstream of a microarray in an illumination light path substantially at a conjugate image plane of the sample, a second automated upstream selective light modulator located upstream of the microarray in the illumination light path substantially at a conjugate image plane of an aperture diaphragm of the objective lens, and a light detector disposed downstream from the microarray in a detection light path substantially at a conjugate image plane of the sample, wherein the first and second selective light modulators and the light detector are operably connected to a suitable controller.
Alternatively, the microarray reader can comprise a first automated downstream selective light modulator located downstream of a microarray in a detection light path substantially at a conjugate image plane of the sample, a second automated downstream selective light modulator located downstream of the microarray in the illumination light path substantially at a conjugate image plane of an aperture diaphragm of the objective lens, and a light detector disposed in the detection light path substantially at a conjugate image plane of the sample and downstream from the first and second downstream selective light modulators and the microarray, wherein the first and second selective light modulators and the light detector are operably connected to a suitable controller.
These and other aspects, features and embodiments of the invention are set forth within this application, including the following Detailed Description and attached drawings. In addition, various references are set forth herein, including in the Cross-Reference To Related Applications, that discuss in more detail certain systems, apparatus, methods and other information; all such references are incorporated herein by reference in their entirety and for all their teachings and disclosures, regardless of where the references may appear in this application.